Choline Oxidase

Abstract
 Choline oxidase is the enzyme that catalyzes the reaction between choline and betaine glycine. Betaine glycine is an osmoprotectant and instrumental in helping plants and bacteria survive dry conditions. Studying the choline oxidase may help in the development of controlling populations of beneficial plants or pathogenic bacteria. The data was analyzed using the BLAST and Rasmol programs. The structure of choline oxidase from Arthrobacter globiformis (bacteria) was compared with the structure of the complimentary protein in Mus musculus (mice), carnitine acetyltransferase (Altschul et al., 2005). There are seven amino acids evolutionarily preserved within the vicinity of the flavin group (between amino acids 460 to 483 of each subunit). Of these seven, three are within 9 Å of the flavin group while the other four are farther away. These three are threonine 463, valine 464, and histadine 466. They are colored blue and are connected with the ligand via white monitor lines. The other four conserved residues are colored cyan, but are too far to really interact with the flavin group (colored cpk). Beta sheets are colored yellow to showcase the secondary structure of the entire subunit. Evolutionary preservation of the Thr463, His 466, and Val 464 may signify an importance in aiding the function of the flavin group as it relates to the activity of the enzyme. Previous studies indicate that His466 is indeed important in the function of choline oxidase (Quaye, Lountos, Fan, Orville, & Gadda, 2008).

Introduction
Choline oxidase is an enzyme which catalyses the chemical reaction choline and 2 molecules of oxygen gas into betaine glycine and two molecules of hydrogen peroxide. Recombinant choline oxidase is a convenient enzyme to introduce into transgenic plants for the synthesis of betaine glycine (Sakamoto and Murata, 2001). Plants adapt to osmotic fluctuations and temperature differences through forming an osmoprotective layer of organic compounds. Betaine Aldehyde, the intermediate of the reaction, is one such osmolyte (Rodwazowski, 1991). Choline oxidase is proposed to use a flavin prosthetic group to assist in the fore-mentioned chemical reaction.

Flavoenzymes are involved in a wide range of biological processes (Joosten and van Berkel, 2007). They have a central role in aerobic metabolism through their ability to catalyze both one- and two-electron transfer reactions. Flavoenzymes are proteins that contain a nucleic acid derivative of riboflavin: the Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN).

Choline oxidase is a catabolic enzyme that belongs to the oxidoreductase family. Oxidoreductases catalyze the transfer of electrons, from electron donating molecules (reductants) to electron accepting molecules (oxidants). The FAD ligand in choline oxidase is an organic cofactor that is covalently bound to the enzyme and essential to the enzyme's function. The FAD ligand is arguably one of the most important parts of the enzyme. Changing some of the residues next to the flavin group may change its stability and have an effect on the rate of enzymatic activity. The most probable residues were mapped.

BLAST analysis
The Basic Local Alignment Search Tool (BLAST) is a web program that aligns a protein with other proteins to find areas of similarities in terms of similar residues. It was used to first find the closest possible sequences to choline oxidase from Arthrobacter globiformis and then to compare the sequence of choline oxidase to caritine acetyltransferase (CRAT) from Mus musculus. Using the National Center for Biotechnology Information (NCBI), acession numbers were obtained for the both proteins. The NCBI website also showed a taxonomic tree for choline oxidase. The only protein from a mammal on that tree was CRAT. For this reason CRAT was chosen for comparison. A protein BLAST was run on just choline oxidase alone. The amino acids sequences near the FAD ligand were noted. Then the accession numbers for both choline oxidase and CRAT were aligned in the protein BLAST for comparison. The E-value predicts the likelihood that two genes are truly similar by random chance. E-values close to zero represent that the correspondence between the two sequences must have arose from similar ancestry and not by random chance. The query in the alignment represents choline oxidase and the subject represents CRAT. The composition of choline oxidase from residues 460 to 483 was of interest since it was close to the flavin group. This is not indicative of the whole structure being similar, but just key amino acids. The BLAST was able to compute the identities, positive and gap scores. Identities represent the number of amino acids were purely conserved between the two sequences. There are seven amino acids evolutionarily preserved within the vicinity of the flavin group. Similar but non-identical residues along with matches were counted for positives. The gap score was zero since neither sequence was shifted by adding a gap in order to align.

RasMol Identification
The RasMol 2.6.5.1 was used to model choline oxidase and the seven evolutionarily preserved amino acids. For this to work, the model of choline oxidase was needed from the protein databank. This file was found, and saved, in the same folder as the RasMol program, as ‘2JBV.spt.’ It was opened in RasMol and the background was changed to white. One subunit of choline oxidase was restricted. The remaining plain subunit was given a backbone of 300 RasMol units and the wireframe of the amino acids residues was turned off. Each RasMol unit is 1/250 of an angstrom. The seven preserved amino acids and the flavin group were space filled with 275 RasMol units and wireframed with 225. The preserved residues were also highlighted in cyan. The three preserved residues closest to the FAD ligand were then changed to a blue color. Monitor lines were drawn between these three residues and the FAD prosthetic group, which was left in the original CPK color. The monitor lines were colored white and spacefilled with 275 RasMol units.

Discussion
Threonine 463, valine 464, and histidine 466 are evolutionarily preserved from bacteria to mice in an enzyme that changed drastically. We found that the Histidine 466 is 4.09 Å from the flavin group, that valine 464 is 5.07 Å away, and that threonine 463 is 7.67 Å away. Each of these three residues should therefore be of some importance to the stability of the flavin group, seeing as they were evolutionarily critical. It is evident that His466 acts as a base in the active site of choline oxidase (Chen and Murata, 2002). However, the functions of Thr463 and Val464 are unknown. Therefore, further research would be necessary to determine the functions of all of the amino acids lining the active site. Mutational studies will also help a great deal in our understanding of the enzyme to see what effect mutating any combination of these three amino acids would have on the enzymatic activity of choline oxidase. Knowing the functions of the amino acids in the active site will enable us to devise genetically modified choline oxidase enzymes. Were any recombinant enzymes found to be functionally better, then they could be introduced to a population of plants to genetically induce potential resistance to drought. On the other hand, recombinant choline oxidase enzymes that were catalytically slower could be studied for their effect on reducing, if not completely suppressing, the growth of pathogenic bacterial colonies.

3D structure of Choline oxidase
2jbv – AgChOx – Arthrobacter globiformis

3nne, 3ljp – AgChOx (mutant)